FlexLISA® Kits

Flexible, Quick and Easy ELISA Development and Optimization!

The Expedeon FlexLISA® kits are designed for the development and optimization of sandwich ELISA assays to detect the presence of any antigen with a high affinity for any antibody pair in complex samples (serum, plasma, urine etc.).

Benefits of the FlexLISA® kit:

Conjugate up to 3 capture and 3 detection antibodies – Ideal for screening

Flexibility to run multiple conjugates on a plate simultaneously

Choice of HRP or AP for enzymatic detection.

A rapid one step protocol is used in this kit, employing pre-blocked microtiter well strips coated with biotin. The FlexLISA® assay is run by adding the sample and the antibody mix to the wells, incubating for one hour, washing and reading the assay plate. FlexLISA® is ideal for quick and reliable ELISA assay development, antibody pair screening and ELISA assay optimization.

Kit components

The kit contains:

Lightning-Link®conjugation technology for the labeling of the capture and detection antibodies of choice

A biotin pre-coated 96 well plate, which can be provided either as clear or black, depending on the enzymatic substrate (12 x 8 strip wells), in a resealable foil pouch.

Not provided: antibodies, assay buffers, enzyme detection solutions.

*Each 10ug vial is enough to run a 96 well plate: less than 0.1ug of capture or detection antibody / well is required and the three reactions provide the flexibility to run multiple conjugates / assays for optimization.

Data

Figure 1. Example of ELISA assay for the detection of CRP using LL-HRP to conjugate the detection antibody and making a calibration curve using CRP standards. Detection reagent: TMB.

Figure 3. Example of ELISA assay for the detection of CRP using LL-AP to conjugate the detection antibody and making a calibration curve using CRP standards. Detection reagent: PNPP. Standard curves fitted with a linear equation (A) and a 4-parameter logistic equation (B). Intra-assay % CV (n=24) have been determined for 800, 200 and 50pg/ml CRP (C).

Figure 4. CRP depleted human serum was spiked with 250pg/ml CRP and serial dilutions of serum were made and loaded to a clear 96 stripwell plate in presence of anti CRP-streptavidin capture antibody and anti CRP-HRP or AP detection antibody. A standard curve using known amounts of CRP was also loaded to the plate. HRP and AP were detected using TMB and PNPP respectively. Once the standard curve had been fitted, the CRP concentrations of the samples added to the plate were determined by interpolating the measured absorbance. For all dilutions, the calculated CRP amount was within 10% the real value.

1. What is different about the FlexLISA® kit?

In a standard ELISA the capture antibody is bound directly to the surface of the well, whereas the 96 stripwell plate provided within the FlexLISA® kit is pre-coated with biotin which binds the capture antibody-streptavidin conjugate with a strong affinity. Therefore, much less capture antibody (40x less) is required compared to standard ELISA assays, saving on costs of expensive antibody.

Unlike other ELISA kits available on the market, the FlexLISA® kit also gives the flexibility of being able to use any antibody of your choice and conjugate as many capture and detection antibodies as required, giving complete flexibility.

Furthermore, the FlexLISA kit contains Expedeon’ Lightning-Link®, the world leading antibody labeling technology, for covalently conjugating your capture and detection antibody. Lightning-Link® kits enable conjugation of any antibody with a free lysine, and each Lightning-Link® kit contains 3 reactions, providing the flexibility to run multiple conjugates at the same time.

2. Are all materials required to run the FlexLISA® included in the kit?

The FlexLISA® kit contains a 96 well plate and conjugation kits to label 3 different capture antibodies and 3 different detection antibodies. Antibodies, assay buffers and enzyme detection solutions are not provided, please see the protocol for additional materials required.

3. How much label and antibody is required for this kit?

The 3x10ug Lightning-Link® labeling kits provided within the FlexLISA® kit will each conjugate 10ug of antibody which is enough to run the 96 well plate. Less than 0.1ug of capture and detection antibody is required per well and the 3 reactions provide the flexibility to run multiple conjugates at once.

4. Do I need to block the plate?

The 96 well stripwell plates are pre-coated with biotin and pre-blocked with blocking agents to prevent high background.

5. What if my sample contains biotin?

The assay can tolerate presence of biotin up to 200pg/ml in the well. If biotin levels in your sample are higher, perform a 1 hour pre-incubation with the Ab mix and then add the sample directly to the plate (no need for washing step).

6. Which substrates do you recommend?

The substrate required depends on the choice of detection label. For HRP the most common substrates are TMB, QuantaRed™ QuantaBlu™, AmpliFlu™ Red, for Alkaline Phosphatase, PNPP, 4-MUP are common place.

7. How should I store my FlexLISA kit?

The kits are shipped at ambient temperature, upon receipt make sure to store at -20°C. All the buffers provided within the kit can be stored at either 4°C or -20°C.

8. How can I avoid high background in my assay?

Please see the below table for possible causes of high background and recommended solutions.

9. I am getting low signal in my assay, what shall I do?

Please see the below table for possible causes of signal and recommended solutions.

10. What should I do about variable replicates?

Please see the below table for possible causes of variable replicates and recommended solutions.

Expedeon is the owner of registered trademark(s) and trademarks which are the subject of pending applications or which are otherwise protected by law. Expedeon trademarks must not be used by you or any other person except where written consent is given.
Expedeon®, Lightning-Link®, InnovaCoat®, Thunder-Link®, Amintra®, RunBlue®, GELFREE®, TruePrime®, and Sunscript®, QualiPhi® and MagniPhi® are registered trademarks of Expedeon Ltd. or its affiliates.
InstantBlue™, AbPureTM, AbSelectTM, BradfordUltraTM, imm-LinkTM, nVoy Stabil-P.A.CTM, PiBindTM, PiColorLockTM, pIMAGOTM, PPS SilentTM, ProteolocTM and CovCheckTM are trademarks of Expedeon Ltd. or its affiliates.
Trademarks of other parties may also appear on this website and in our documents, and are the property of their respective owners.